Development, Use, and Validation of (Q)SARs for Predicting Genotoxicity and Carcinogenicity: Experiences from Italian National Institute of Health Activities.

In silico approaches are now increasingly accepted in several areas of toxicology to rapidly assess chemical hazard without the need for animal testing. Among in silico tools, quantitative and qualitative structure-activity approaches ((Q)SARs) are the most typically applied methods to predict hazard in the absence of experimental data. This paper provides an overview of different protocols that can be applied while dealing with (Q)SARs in different scenarios, namely, (Q)SAR development, use, and validation. Examples of protocols adopted in the three scenarios are reported, derived from the authors' experience in working at the Predictive Toxicology unit of the Italian National Institute of Health, focusing on the endpoints of carcinogenicity and genotoxicity.The illustrated activities are in line with the Institute's mission, the main center of research, control, and technical-scientific advice on public health in Italy.

A comprehensive toxicological analysis of panel of unregulated e-cigarettes to human health.

Electronic cigarettes, commonly referred to as e-cigarettes have gained popularity over recent years especially among young individuals. In the light of the escalating prevalence of the use of these products and their potential for long-term health effects, in this study as the first of its kind a comprehensive toxicological profiling of the liquid from a panel of unregulated e-cigarettes seized in the UK was undertaken using an in vitro co-culture model of the upper airways. The data showed that e-cigarettes caused a dose dependent increase in cell death and inflammation manifested by enhanced release of IL1ß and IL6. Furthermore, the e-cigarettes induced oxidative stress as demonstrated by a reduction of intracellular glutathione and an increase in generation of reactive oxygen species. Moreover, the assessment of genotoxicity showed significant DNA strand breaks (following exposure to Tigerblood flavoured e-cigarette). Moreover, relevant to the toxicological observations, was the detection of varying and frequently high levels of hazardous metals including cadmium, copper, nickel and lead. This study highlights the importance of active and ongoing collaborations between academia, governmental organisations and policy makers (Trading standards, Public Health) and national health service in tackling vape addiction and better informing the general public regarding the risks associated with e-cigarette usage.

In vitro study of the UV-filter homosalate effects on rat and human thyroid cells.

Homosalate is a UV-B filter, commonly used in sunscreens and personal-care products. Homosalate was shown to exert estrogenic and anti-androgenic effects in animal models, while few data are available on the effects of Homosalate on thyroid cells. The aim of this study was to evaluate if Homosalate exposure could exert adverse effect on thyroid cells in vitro. FRTL-5 and NHT were treated with increasing concentration of Homosalate for 24-48-72 h. Cell viability was assessed by WST-1. Cell proliferation was evaluated by cristal violet. Micronucleus staining was performed to assess genotoxicity. mRNA levels of thyroid-related genes (TSHR, TPO, TG, NIS, and PAX8) were evaluated by RT-PCR. Changes in ROS production by FRTL-5 and NHT were assessed with H2DCFDA. Homosalate significantly reduced cell viability after 72 h in FRTL-5 starting from the concentration 250 µM, while in NHT, Homosalate exposure significantly reduced cell viability after 48 and 72 h only at highest concentration (2000 µM). Cell proliferation was not modified by Homosalate at any concentration and time-point. Homosalate significantly up-regulated mRNA expression levels of TPO and Tg genes in FRTL-5, while a significant increase only in Tg mRNA expression was observed in NHT. No changes in ROS production was found in both cell types. The present study suggest that the effects of Homosalate exposure may differ according to the type of cell tested. The in vitro exposure of thyroid cells to Homosalate produces: i) cytotoxicity at high concentrations or after long time of incubation, ii) genotoxicity only in rat thyroid cells at the highest concentration, iii) upregulation of Tg mRNA in both thyroid cell types and of TPO mRNA in rat thyroid cells, iv) no changes in cell proliferation or oxidative stress. Further studies on the effects of Homosalate on thyroid cells should be encouraged.

Scoping review on the genotoxicity of silver nanoparticles in endodontics: therapeutic saviors or genetic saboteurs?

Silver nanoparticles (AgNPs) have gained prominence in endodontics due to their exceptional antimicrobial properties. However, concerns regarding their genotoxic potential have prompted investigations into their safety profiles. This scoping review aims to consolidate current knowledge on the genotoxic effects of AgNPs in the field of endodontics. A thorough literature search across seven electronic databases was conducted using specific keywords. Inclusion criteria included experimental studies published in English from January 1960 to March 2024, addressing the genotoxicity of AgNPs in endodontic applications. Study selection and data extraction were conducted independently. The Quality Assessment Tool For In Vitro Studies (QUIN) tool was employed to evaluate the risk of bias in each study. 5 articles were selected, of which 3 were in vitro experimental designs, while the remaining were ex vivo studies. All were published between 2009 and 2021. AgNPs have been used as root canal irrigating solutions, pulp-capping materials, and root canal sealers. Most studies employed the comet assay for genotoxic evaluation. One study was found to have a low risk of bias, while others were categorized as having a medium risk. Mixed findings were noted on the genotoxic effects of AgNPs using various assays. The genotoxic potential of AgNPs somewhat poses concerns for endodontic practices. This review highlights the need for further research to develop safer alternatives and optimize their concentrations and exposure durations.

In vivo micronucleus assay on sodium molybdate in rats and its impact on the overall assessment of the genotoxicity of molybdenum substances.

In this paper we present methodological and experimental details and results from an OECD Test Guideline 474 and GLP-compliant in vivo micronucleus study on sodium molybdate dihydrate in Sprague Dawley rats. Prior to the conduct of this study, there was a data-gap for reliable in vivo genotoxicity data for molybdenum substances. The presentation of the new study is complemented by a review of other available in vitro and in vivo data on the genotoxicity of molybdenum substances, focussing on substances where the contained or released molybdate ion, MoO42-, is considered the responsible moiety for any toxicological effect (grouping/category approach). After consideration of the relevance and reliability of all available data, the absence of a concern for genotoxicity of molybdate in vitro and in vivo is concluded.

Identification and in vitro genotoxicity assessment of forced degradation products of glimepiride and glyburide using HEK cell-based COMET assay.

This study focuses on characterizing the forced degradation products of antidiabetic drugs glimepiride (GMD) and glyburide (GBD), with previously unexplored genotoxicity. Drugs underwent stress induced by acid, base, and hydrogen peroxide. For GMD, impurities were profiled and isolated using Hypersil Gold C8 (250 × 10 mm, 5 µ) through semi-preparative HPLC with a fraction collector. For GBD, impurity profiling was performed using semi-preparative HPLC (Hypersil GOLD C18, 250 × 10 mm, 5 µ), and reverse-phase flash chromatography (FP ECOFLEX C18 4 g column) for isolation. Although five GMD and three GBD impurities were detected, only three GMD and two GBD impurities were separated and assessed for purity using analytical RP-HPLC with the purity percentages ranging from 96.6% to 99.9%. LC-Orbitrap MS was used to identify these three GMD impurities (m/z: 408.122, 338.340, 381.160) and two GBD impurities (m/z: 369.065, 325.283). ProTox-II in silico predictions classified all impurities as class 4 and 5, with no positive genotoxicity indications. In vitro comet assays, using HEK cells, indicated that for GMD, impurity 2 and impurity 5 were less genotoxic, whereas impurity 4 exhibited genotoxicity. For GBD, both impurities 1 and 3 were found to be genotoxic, with impurity 3 showing a higher level of genotoxicity than impurity 1.

Do professional painters comprise a high risk group for genotoxicity? A systematic review.

Professional painters represent an occupational population group that deserves attention for study in the field of occupational toxicology due to the wide range of complex chemical mixtures they are exposed to. It is imperative to underscore that the International Agency for Research on Cancer has classified commercial painting as a high-risk occupation for the development of cancer. Given this context, the primary objective of the present study was to conduct a systematic review aimed at addressing the following question: are car painters at occupational risk regarding potential genotoxicity? To address this question, a selection process was undertaken, with three reviewers carefully selecting, reading, and analyzing full manuscripts from 26 studies included in this review. The technical rigor of these studies underwent meticulous scrutiny, culminating in the classification of six studies as Strong, eight as Moderate, and 12 as Weak, predicated on the extent of confounders considered. Taken together, the findings suggest that chemical substances from paints may indeed pose a risk of genotoxicity for professionals in this field, as all studies indicated genotoxicity among professional painters through various tests.

Assessing nanotoxicity of food-relevant particles: A comparative analysis of cellular responses in cell monolayers versus 3D gut epithelial cultures.

Engineered nanoparticles (NPs) are extensively used in the food industry, yet safety concerns remain. The lack of validated methodologies is a bottleneck towards resolving this uncertainty. Hence, the current study aims to compare two cell models by examining the toxicological impacts of two food-relevant NPs (SiO2 and Ag) on intestinal epithelia using monolayer Caco-2 cells and full-thickness 3D tissue models of human small intestines (EpiIntestinal™). Comprehensive characterization and dosimetric analysis of the NPs were performed to determine effective doses and model realistic exposures. Neither genotoxicity nor cytotoxicity were detected in the 3D tissues after NP treatment, while the 2D cultures exhibited cytotoxic response from Ag NP treatment for 24 hr at 1 µg/ml. Hyperspectral imaging and transmission electron microscopy confirmed uptake of both NPs by cells in both 2D and 3D culture models. Ag NPs caused an increase in autophagy, whereas SiO2 NPs induced increased cytoplasmic vacuolization. Based on realistic exposure levels studied, the 3D small intestinal tissue model was found to be more resilient to NP treatment compared to 2D cell monolayers. This comparative approach towards toxicological assessment of food relevant NPs could be used as a framework for future analysis of NP behavior and nanotoxicity in the gut.

Edible vegetables grown in the vicinity of electronic wastes: A study of potential health risks and DNA damage in consumers.

There has been a growing concern on the health effect of edible plants growing near/on/within the vicinity of dumpsites. This study investigated two edible vegetables: Amarathus hybridus and Talinum triangulare (Jacq) grown in the vicinity of a major informal dumpsite of electronic waste in Nigeria. The levels of polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), and heavy metal concentrations in the vegetables were measured. The health risks of consuming the vegetables were assessed using the hazard index (HI), lifetime cancer risk (LCR), estimated daily intake (EDI), and hazard quotient (HQ). Using the Ames Salmonella fluctuation test on Salmonella typhimurium (TA100 and TA98) and the SOS chromo test on Escherichia coli (PQ37), the mutagenicity and genotoxicity of the vegetables were evaluated. The two vegetables have elevated levels of heavy metals, PBDEs, PCBs, and hazardous PAHs. Compared to A. hybridus, T. triangulare was more contaminated. The amounts of organic constituents and heavy metals in the vegetables correlated favorably. The levels of the HQ, HI, and LCR were above the suggested guideline values, indicating a significant risk of both carcinogenic and non-carcinogenic consequences, particularly in children. The two vegetables were mutagenic even at 50 % concentration in the Ames test. This was corroborated with SOS-chromo test results showing that the two vegetables were indeed genotoxic. This study demonstrated the harmful effects of growing food crops close to dumpsites; therefore, sufficient measures should be implemented to stop farmers and individuals from utilizing dirt from dumps as fertilizer or from planting in soil that has been used as a dump in the past or present.

Bridging toxicological properties of environmental chemicals between animals and humans using healthy organoid systems.

The application of organoids derived from animal tissues and human-induced pluripotent stem cells to safety assessments of environmental chemicals has been introduced over the last decade. One of the objectives of this approach is to develop an alternative method for animal toxicological studies, while another is to focus on the local reactions of chemicals in each organ/tissue. One of the most important goals is bridging the toxicological properties of chemicals between animals and humans, which may be compared on a level playing field using healthy organoids derived from both animals and humans in vitro, excluding species difference in the absorption, distribution, metabolism, and excretion properties of chemicals in vivo. An overview of the application of organoid systems to safety assessments of environmental chemicals, including general toxicology, developmental toxicology, carcinogenicity, and mutagenicity, was provided herein, and bridging strategies using both animal and human organoids are proposed as a future perspective.

Does waterpipe smoke induce genotoxicity (DNA damage) in mammalian cells in vivo? A systematic review.

The waterpipe works by placing tobacco in a bowl with holes at the bottom, which is connected to a tube leading to a water-filled container. Upon heating the tobacco product with hot charcoal placed atop it, the emanating smoke is inhaled by the user via a hose linked to the water receptacle. The aim of this literature review is to evaluate whether the use of waterpipes can indeed induce genotoxicity in mammalian cells in vivo. Additionally, the study aims to assess the quality of the included research articles on this topic to ensure the reliability of the findings. We performed comprehensive searches in PubMed, SCOPUS, and Web of Science to identify relevant articles published until July 2024. The findings confirmed that waterpipe smoke induces genetic damage. This assertion is supported by the fact that 11 studies (out of 15) received a Strong or Moderate assessment categorization, suggesting that the majority of studies adhered to most technical standards, thereby enhancing the reliability of the research findings. Regarding the types of DNA damage reported, DNA strand breaks, chromosome damage and oxidative DNA damage were found in this review. Taken together, this study holds significant importance in assessing the efficacy of genotoxicity assays in detecting DNA damage due to waterpipe smoke and the comet and micronucleus assays are suitable biomarkers for biomonitoring people who use waterpipe.

Assessing Genotoxicity and Cytotoxicity Induced by X-Ray Exposure From Cone Beam Computed Tomography at Varied Fields of View.

INTRODUCTION: The practice of dentistry benefits greatly from cone beam computed tomography (CBCT) and advantages should be prioritized over hazards; even modest doses of X-rays have the potential to have cytotoxic effects, damage DNA through their clastogenic impact, and stimulate the creation of micronuclei along with further nuclear changes. AIMS AND OBJECTIVES: To assess the genotoxicity and cytotoxicity of X-rays in exfoliated oral mucosal cells from patients who underwent CBCT scans at different fields of view (FOV), and to examine and assess the extent of cytotoxicity and genotoxicity caused by X-rays in oral exfoliated cells of people who were subjected to CBCT at different fields of view (FOV). MATERIAL AND METHODS: Following CBCT exposure, 66 patients were chosen from the Department of Oral Medicine and Radiology at the SVS Institute of Dental Sciences, Mahbubnagar. Cells from the buccal mucosa were then extracted using the exfoliative cytology method, and the samples were examined under a microscope to look for nuclear and cytological abnormalities. RESULTS: A paired t-test analysis revealed that mean micronuclei increased significantly in each study group from before to after exposure. It increased in Group I from 93.59 to 96.05, in Group II from 83.27 to 91.86, and in Group III from 86.05 to 97.00. Various test analyses revealed an important relation between exposure status and the presence of karyorrhexis in Group III. There was no association in other groups. CONCLUSION: The study revealed a significant increase of micronuclei in subjects after exposure to radiation at various FOVs. There was an increased karyorrhexis following radiation exposure in all groups at various FOVs. The significant association between exposure and karyorrhexis in the larger size FOV group was noticed further potentiating the extent of increased damage as the size of FOV is increased.

Biosynthesis and characterization of gold nanoparticles from citrullus colocynthis (L.) schrad pulp ethanolic extract: Their cytotoxic, genotoxic, apoptotic, and antioxidant activities.

The age-old discipline of plant therapy has gained renewed importance through the utilization of plants for the synthesis of metal nanoparticles. However, toxicity testing and characterization of the recently synthesized nanomaterials are essential to evaluating their appropriate application. Citrullus colocynthis is a medicinal plant with several health benefits. Herein, we used its ethanolic pulp extract (PE) to manufacture gold nanoparticles (PE-AuNPs). Various approaches were employed to assess the MTT50 and NR50 values of PE and PE-AuNPs at different concentrations in the human hepatocarcinoma cell line (HepG2). The study aimed to assess the genotoxic effects and in vivo toxicity of PE and PE-AuNPs at MTT50 dosages. The quasi-spherical, cubic/triangular prisms, and nail-looking particles exhibited no antioxidant properties. They had an absorbance peak between 540 and 560 nm, diameters of less than 20 nm, hydrodynamic diameters of 177.9 nm, and a negative surface charge (-10.3 mV). The significant role of plant phytochemicals in the formation of metal nanoparticles is confirmed by the diminished antioxidant capacity of extract residues following PE-AuNP synthesis. PE-AuNPs exhibited in vivo and cytotoxic effects at relatively lower concentrations compared to PE. In contrast to PE, PE-AuNPs exhibited lower genotoxic at MTT50 dosages. Despite having MTT50 values of approximately 1.95 ± 0.06 and 0.89 ± 0.03 mg/ml, PE and PE-AuNPs can still be considered biocompatible. Nonetheless, our results suggest that the characteristics of recently produced nanoparticles can differ from those of the matching plant. Further investigation can provide a better understanding of the possible therapeutic and pharmacological impacts of PE-AuNPs.

Bifidobacterium longum subsp. infantis YLGB-1496-Toxicological evaluation.

Bifidobacterium infantis YLGB-1496, originally isolated from breast milk from a Taiwanese mother, is under study for use as a probiotic. As part of safety assessment, an Ames, in vivo mouse micronucleus, and in vivo mouse spermatocyte chromosome aberration assay were conducted along with a 13-week oral rat toxicity study. B. infantis YLGB-1496 had no activity in any of the genotoxicity assays. Administration of the bacteria to Sprague-Dawley rats at doses ranging from 0 to 1.5 g/kg bw/day had no treatment-related effects on any of the endpoints measured. There appear to be no concerns for translocation or pathogenicity of B. infantis YLGB-1496 based on extensive experience with the species in general. The results of the current investigations support potential use of B. infantis YLGB-1496 as a probiotic in infant formula.

Solubilizer of bacterial origin surfactin increases the biological activity of C60 fullerene.

Currently, there exists conflicting data regarding the biological activity of unmodified fullerene C60. Various sources report its toxicity, geroprotective activity, and potential interaction with DNA. Contradictory findings regarding the toxicity of C60 may arise from the use of toxic solvents, as well as the influence of bioavailability and bioactivity on the preparation conditions of C60 suspensions. Furthermore, the microbiota of experimental animals can impact geroprotective activity results by releasing surfactants that facilitate substance penetration through the cell membrane. In this study, we selected conditions for solubilizing fullerene C60 in a solution of surfactin, a surfactant of bacterial origin, as well as in a 2% aqueous solution of TWEEN 80, employing ultrasound. Through bioluminescent analysis using lux biosensors in Escherichia coli MG1655, we observed that C60 in surfactin reduced induced genotoxic and oxidative stress. Given that surfactin enhances membrane permeability to fullerene C60, suspensions of fullerene in designated concentrations of surfactin can be regarded as a DNA protector and antioxidant, warranting further investigation as a promising component of novel drugs.

Exploring the toxicity profile of coriander (C. sativum L.) essential oil: implications for translational toxicological research.

The plant species C. sativum L. is a staple in cuisine and holds significant ethnopharmacological value. Its essential oil (EO) is of particular interest, yet its toxicity profile remains a subject of inquiry. This study aimed to elucidate the chemical constituents of C. sativum L. EO and evaluate its toxicity through various parameters, including cytotoxicity assays on HaCaT keratinocytes, in vivo toxicity tests on Galleria mellonella larvae, in vivo genotoxicity assessments on mice and cytotoxicity assays on human erythrocytes. Notably, major constituents such as 2-decen-1-ol, dec-(2E)-enal, and 1,6-octadien-3-ol were found to remain predominant. The IC50 value for the essential oil on the keratinocyte cell line was determined to be 60.13 ± 2.02 µg/mL. However, in vivo toxicity tests with G. mellonella larvae demonstrated safety at doses below 4.5 g/kg. Additionally, genotoxicity assessment revealed that a single dose of 20 mg/mL (5 mg/kg) did not induce a significant increase in micronuclei formation. EO concentrations above 250 µg/mL led to significant changes in human erythrocytes cell viability (p < 0.0001), resulting in over 60% hemolysis. These findings collectively suggest that the essential oil of C. sativum L. exhibits a suitable toxicity profile for conducting preclinical studies in vertebrate animal models.

Synergistic Mitochondrial Genotoxicity of Carbon Dots and Arsenate in Earthworms Eisenia fetida across Generations: The Critical Role of Binding.

The escalating utilization of carbon dots (CDs) in agriculture raises ecological concerns. However, their combined toxicity with arsenic remains poorly understood. Herein, we investigated the combined mitochondrial genotoxicity of CDs and arsenate at environmentally relevant concentrations across successive earthworm generations. Iron-doped CDs (CDs-Fe) strongly bound to arsenate and arsenite, while nitrogen-doped CDs (CDs-N) exhibited weaker binding. Both CDs enhanced arsenate bioaccumulation without affecting its biotransformation, with most arsenate being reduced to arsenite. CDs-Fe generated significantly more reactive oxygen species than did CDs-N, causing stronger mitochondrial DNA (mtDNA) damage. Arsenate further exacerbated the oxidative mtDNA damage induced by CDs-N, as evidenced by increased reactive oxygen species, elevated 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-OHdG) levels, and a higher correlation between 8-OHdG and mtDNA damage. This was due to arsenic inhibiting the antioxidant enzyme catalase. This exacerbation was negligible with CDs-Fe because their strong binding with arsenic prevented catalase inhibition. Maternal mitochondrial DNA damage was inherited by filial earthworms, which experienced significant weight loss in coexposure groups coupled with mtDNA toxicity. This study reveals the synergistic genotoxicity of CDs and arsenate, suggesting that CDs could disrupt the arsenic biogeochemical cycle, increase arsenate risk to terrestrial animals, and influence ecosystem stability and health through multigenerational impacts.

Methodological steps forward in toxicological in vitro screening of mineral wools in primary rat alveolar macrophages and normal rat mesothelial NRM2 cells.

Man-made vitreous fibers (MMVF) comprise diverse materials for thermal and acoustic insulation, including stone wool. Depending on dimension, durability, and dose, MMVF might induce adverse health effects. Therefore, early predictive in vitro (geno)toxicity screening of new MMVF is highly desired to ensure safety for exposed workers and consumers. Here, we investigated, as a starting point, critical in vitro screening determinants and pitfalls using primary rat alveolar macrophages (AM) and normal rat mesothelial cells (NRM2). A stone wool fiber (RIF56008) served as an exemplary MMVF (fibrous vs. ground to estimate impact of fiber shape) and long amosite (asbestos) as insoluble fiber reference. Materials were comprehensively characterized, and in vivo-relevant in vitro concentrations defined, based on different approaches (low to supposed overload: 0.5, 5 and 50 µg/cm2). After 4-48 h of incubation, certain readouts were analyzed and material uptake was investigated by light and fluorescence-coupled darkfield microscopy. DNA-strand break induction was not morphology-dependent and nearly absent in both cell types. However, NRM2 demonstrated material-, morphology- and concentration-dependent membrane damage, CINC-1 release, reduction in cell count, and induction of binucleated cells (asbestos > RIF56008 > RIF56008 ground). In contrast to NRM2, asbestos was nearly inactive in AM, with CINC-1 release solely induced by RIF56008. In conclusion, to define an MMVF-adapted, predictive in vitro (geno)toxicity screening tool, references, endpoints, and concentrations should be carefully chosen, based on in vivo relevance, and sensitivity and specificity of the chosen cell model. Next, further endpoints should be evaluated, ideally with validation by in vivo data regarding their predictivity.

Epigenotoxicity: Decoding the epigenetic imprints of genotoxic agents and their implications for regulatory genetic toxicology.

Regulatory genetic toxicology focuses on DNA damage and subsequent gene mutations. However, genotoxic agents can also affect epigenetic marks, and incorporation of epigenetic data into the regulatory framework may thus enhance the accuracy of risk assessment. Additionally, epigenetic alterations may identify non-genotoxic carcinogens that are not captured with the current battery of tests. Epigenetic alterations could also explain long-term consequences and potential transgenerational effects in the absence of DNA mutations. Therefore, at the 2022 International Workshops on Genotoxicity Testing (IWGT) in Ottawa (Ontario, Canada), an expert workgroup explored whether including epigenetic endpoints would improve regulatory genetic toxicology. Here we summarize the presentations and the discussions on technical advancements in assessing epigenetics, how the assessment of epigenetics can enhance strategies for detecting genotoxic and non-genotoxic carcinogens and the correlation between epigenetic alterations with other relevant apical endpoints.

Mutagenicity of the agriculture pesticide chlorothalonil assessed by somatic mutation and recombination test in Drosophila melanogaster.

Chlorothalonil (CTL) is a pesticide widely used in Brazil, yet its mutagenic potential is not fully determined. Thus, we assessed the mutagenicity of CTL and its bioactivation metabolites using the somatic mutation and recombination test (SMART) in Drosophila melanogaster, by exposing individuals, with basal and high bioactivation capacities (standard and high bioactivation cross offspring, respectively), from third instar larval to early adult fly stages, to CTL-contaminated substrate (0.25, 1, 10 or 20 µM). This substrate served as food and as physical medium. Increased frequency of large single spots in standard cross flies' wings exposed to 0.25 µM indicates that, if CTL is genotoxic, it may affect Drosophila at early life stages. Since the total spot frequency did not change, CTL cannot be considered mutagenic in SMART. The same long-term exposure design was performed to test whether CTL induces oxidative imbalance in flies with basal (wild-type, WT) or high bioactivation (ORR strain) levels. CTL did not alter reactive oxygen species and antioxidant capacity against peroxyl radicals levels in adult flies. However, lipid peroxidation (LPO) levels were increased in WT male flies exposed to 1 µM CTL. SMART and LPO alterations were observed only in flies with basal bioactivation levels, pointing to direct CTL toxicity to DNA and lipids. Survival, emergence and locomotor behavior were not affected, indicating no bias due to lethality, developmental and behavioral impairment. We suggest that, if related to CTL exposure, DNA and lipid damages may be residual damage of earlier life stages of D. melanogaster.